The present invention relates to the identification and characterization of novel DNA sequences that are specific to Listeria monocytogenes strains that are commonly associated with human disease and provides improved oligonucleotides and methods for their use in the detection and typing of these strains.
Listeria monocytogenes, a bacterium, is the causative agent of listeriosis, a serious disease of humans and animals that can be transmitted by means of contaminated food. Newborns, the elderly, and immunocompromised individuals are especially prone to infection. Listeria are commonly found in the environment and, unlike most other human pathogens, are capable of growth at refrigeration temperatures, often leading to problematic contamination of cold-stored foods. Such foods, especially dairy products, have been implicated in numerous cases of sporadic listeriosis as well as several common-source epidemics of the disease.
Even though numerous serotypes of L. monocytogenes have been identified with the antigenic scheme of Seeliger and Hxc3x6hne (Methods Microbiol. 13:31-49 (1979)), three serotypes (1/2a, 1/2b, and 4b) account for the vast majority of clinical isolates. Furthermore, strains of serotype 4b have been implicated in a large fraction (ca. 40%) of sporadic listeriosis cases and virtually all common-source outbreaks reported in Europe and North America during the past 20 years (Schuchat et al. 1991. Clin. Microbiol. Rev. 4:169-183). Pulsed-field fingerprinting of chromosomal DNA has revealed that serotypes 4b, 4d, and 4e strains constitute one genomic subdivision (cluster IIB) (Brosh et al. 1994. Appl. Environ. Microbiol. 60:2584-2592). From the perspective of human disease, serotype 4b strains are the major component of this genomic cluster because serotype 4d and 4e strains are isolated only rarely from foods and virtually never from patients (Farber et al., 1991. Microbiol. Rev. 55:476-511).
Serologic methods currently employed to type strains of Listeria monocytogenes requires culturing and isolating the bacteria and highly specific antisera. These techniques are performed effectively only in a small number of reference laboratories (Seeliger and Hohne. 1979. Methods Microbiol. 13:31-49). Monoclonal antibodies have been described that are specific for individual serotypes; however, because monoclonal antibodies are highly specific, subtle variations in epitope structure can render them non-reactive. Existing hybridization and PCR-based methodologies employing oligonucleotides have been described to identify Listeria monocytogenes but these methods can not be used to identify individual strains and serotypes most commonly associated with human disease because the nucleic acids targeted by the oligonucleotides are shared across serotypes. As described above, pulse-field fingerprinting has been used to identify L. monocytogenes genomic divisions, which have been correlated with individual serovars and subgroups but this technique is not amenable to routine analysis because it requires expensive equipment to run the assay and analyze the data, and is labor intensive. It would, therefore, be useful to identify and characterize genetic and molecular features unique to L. monocytogenes serotypes to facilitate the development of methods and reagents for molecular subtyping and detection of strains most commonly associated with human disease.
The present invention provides compositions and methods for detecting and identifying Listeria monocytogenes genomic cluster IIB strains which are commonly associated with human disease.
In one embodiment the invention provides an isolated nucleic acid of L. monocytogenes genomic DNA that is unique to genomic cluster IIB strains.
In one aspect, the isolated nucleic acid comprises DNA having preferably at least about 80% sequence identity, and most preferably at least about 90% sequence identity to genomic cluster IIB DNA or its complement. In another aspect, the invention provides recombinant nucleic acids that hybridize to genomic cluster IIB-DNA. Preferably, hybridization occurs under stringent hybridization and wash conditions.
In another aspect the invention provides an isolated nucleic acid molecule comprising a DNA encoding one or more polypeptide(s) involved in the expression of cluster IIB polypeptides or its complement. The invention further provides antibody which specifically binds to a genomic cluster IIB polypeptide.
In a still further aspect the invention provides a vector comprising an isolated nucleic acid comprising genomic cluster IIB-DNA or its variants.
In yet another aspect the invention provides methods for detecting L. monocytogenes genomic cluster IIB in a composition of matter comprising cluster IIB specific nucleotide sequences. In a further aspect of the invention, oligonucleotide probes derived from the aforemention sequences and methods for their synthesis, labeling and use in the detection and identification of L. monocytogenes cluster IIB strains are provided. In a broad embodiment, the methods comprise contacting the nucleic acids of L. monocytogenes with one or more oligonucleotides under conditions permitting hybridization and detecting the hybridized oligonucleotide.